GOAL #1 LMB-100 Combinations. LMB-100 (previously RG7787) is a next generation recombinant immunotoxin (RIT) developed by the Pastan Lab (NCI/ LMB) in collaboration with Roche. This RIT binds to the cancer antigen MSLN delivers a potent bacterial toxin to the cell cytosol. The toxin, a molecularly engineered variant of Pseudomonas exotoxin A, kills cells by irreversibly modifying a critical enzyme in the protein synthesis pathway resulting in a halt in the cell's ability to produce new proteins. This insult triggers apoptosis in many cell types. AIM 1 (on-going): Evaluate efficacy of MSLN-targeted RITs in pancreatic cancer patients. At least 70% of pancreatic adenocarcinomas express the surface antigen MSLN, making these tumors good targets for MSLN-targeted therapies. Based on our previous results, we initiated A Phase Ib/II Study of Mesothelin-Targeted Immunotoxin LMB-100 in Combination with Nab-Paclitaxel in Participants with Previously Treated Metastatic and/ or Locally Advanced Pancreatic Ductal Adenocarcinoma (PI Alewine) to determine the safety, tolerability and efficacy of the LMB-100 + NAB-paclitaxel combination. This trial received IRB approval in 6/2016 and formally opened for accrual 8/2016. Arm A1 (Phase 1 examining short infusion LMB-100 with nab-paclitaxel) has been completed after accrual of 13 patients. A manuscript is currently in preparation. Accrual to Arm A2 (Phase 2 of same combination) has begun. Arm B1 (Phase 1 examining long infusion LMB-100) has accrued 15 patients and MTD has been determined. AIM 2 (ongoing): Determine whether RG7787 or other mesothelin-targeted RITs can boost the effect of immune activating drugs like immune checkpoint inhibitors (ICIs). Pancreatic adenocarcinoma produces an immunosuppressive microenvironment. Killing tumor cells with oncolytic viruses or administering anti-tumor vaccines can cause immune activation. Combining these treatments with ICIs has been demonstrated to produce anti-tumor immune responses in pre-clinical models of pancreatic cancer. We hypothesize that since RG7787 uses a bacterial toxin to kill tumor cells, it may also induce immune activation within the pancreatic cancer microenvironment that could be leveraged to induce an anti-tumor immune response in combination with ICIs. Because our immunotoxins bind only to human (hMSLN) and not to native mouse MSLN (mMSLN), completing this project requires development of a syngeneic mouse pancreatic cancer cell line expressing hMSLN into a mouse strain that will not reject these cells. To produce such models, we initiated a collaboration with the CAPR group at Frederick (Serguei Kozlov, Leidos) to develop new mouse models with express hMSLN. During FY2018 we performed characterization of 2 such transgenic mouse lines, as well as a syngeneic cell line expressing hMSLN. We are continuing characterization of a third model. With these models in place, anti-tumor experiments using combined ICI and immunotoxin treatment can begin in FY19. AIM3 (new): Determine the effect of mesothelin-targeted RIT on tumor cell protein synthesis. RIT inhibits new protein synthesis. The effect of this on individual levels of tumor cell secreted proteins within tumor cells and in the tumor microenvironment has not been previously examined. We used existing athymic nude mouse xenograft model of pancreatic cancer, as well as a new syngeneic model using a cell line co-developed with CAPR to examine this effect. We found that RIT decreases levels of short-lived proteins including many cancer-associated proteins, and tumor secreted factors important in tumorigenic signaling. This data was presented at AACR Annual Meeting 2018. A manuscript is currently in preparation. AIM 4 (ongoing): Determine whether delivery and efficacy of RG7787 to tumors can be improved by co-administration of an anti-fibrotic drug. Pancreatic adenocarcinoma contains a large volume of stroma that inhibits delivery of anti-cancer therapeutics, provides cytokines that help tumor cells survive under stress, and suppressed anti-tumor immune activation. We have entered into a collaboration to test an anti-fibrotic/ anti-angiogenesis drug developed by a extramural investigator Zhi-ren Liu. Our resubmitted Intramural/ Extramural Grant application was not approved FY2017, but the project is now the subject of a Bench To Bedside Award application. GOAL #2 Understanding MSLN Signaling. MSLN is the target of many therapeutics being tested in clinical trials. It has previously been shown to increase the aggressiveness of pancreatic cancer. It is unknown whether current anti-MSLN therapies inhibit the pro-tumorigenic signaling by MSLN or whether they are just addressing toxic payloads to tumor cells. AIM 1 (ongoing): Determine a phenotype of MSLN loss in pancreatic cancer cells. We deleted MSLN from a human pancreatic cancer cell line using CRISPR-Cas9 gene editing. We found that these cells grow poorly in the abdominal cavity of mice. This data was presented at AACR Annual Meeting 2018. A manuscript reporting this finding is currently in preparation. We are continuing to characterize the signaling pathways responsible. AIM 2 (ongoing): Determine why MSLN can be detected in the blood of some but not all patients with tumors that make MSLN. We have identified a model system in mice in which to investigate this question. We are continuing to identify the mechanism responsible for the differential secretion of MSLN into blood.